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Vetralla, Massimo (2018) Development of a microfluidic approach for the real-time analysis of autocrine and paracrine TGF-beta signaling. [Ph.D. thesis]

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Abstract (english)

Transforming Growth factor-beta (TGF-beta) is a multifunctional cytokine that controls several cellular processes including cell proliferation, differentiation, migration and apoptosis. Despite TGF-beta mediates a variety of biological processes, the signaling is spread in a relatively simple manner. TGF-beta is secreted as a latent complex and requires an enzymatic activation to bind its receptor and promote intracellular signaling. Temporal control of TGF-beta signaling is essential to regulate embryogenesis and also to maintain homeostasis in mature tissue. Pathway dysregulation is associated to pathological conditions, such as fibrosis and carcinogenesis. The first part of this work is focalized on TGF-beta signaling pathway dynamics. By exposing cells integrated in a microfluidic platform to multiple, periodic administrations of TGF-beta, it was identified how different TGF-beta stimulations are associated to different pathway dynamics and expressions of target genes. In particular the analysis is focused on SMAD7 protein, responsible of pathway inactivation and it was demonstrated that SMAD7 expression is indipendent from the frequency of TGF-beta stimulation.
Different clinically relevant outcomes can be obtained by controlling TGF-beta signaling pathway activation, duration and amplitude via intracellular and extracellular mechanisms, thus, targeting the TGF-beta signaling in the
pharmacological treatment of liver diseases constitute a novel approach. In the second part of this work, taking advantage of the microfluidic technology, a novel device to recapitulate in vitro the main features of hepatic fibrosis was created. In this microfluidic platform, the confined microenvironment promotes the accumulation of profibrotic or antifibrotic factors in response to profibrotic or antifibrotic stimuli, secreted by different kinds of cells involved in hepatic fibrosis. Moreover, the production, activation and function of endogenous TGF-beta from cells in the hepatic microenvironment was analysed, with specific focus on hepatic stellate cells and macrophages that are respectively responsible of the
fibrotic and inflammatory process. It was also analysed the effect of
Thrombospondin-1, a specific extracellular matrix glycoprotein, in the activation of the TGF-beta latent complex. Thrombospondin-1 is found to be increased in the serum of fibrotic patients and for this reason, the effect of thrombospondin-1 in the activation of autocrine TGF-beta secreted from hepatic stellate cells was evaluated. Finally it was analysed the collagen production mediated by TGF-beta in hepatic cells and stellate cells, revealing that the latter produce collagen in response to endogenous or exogenous TGF-beta. The third part of the work involved the development of a human inducible Pluripotent Stem Cells (iPSCs) derived hepatocyte model, using microfluidic platforms. The differentiation protocol was applied to obtain functional hepaticlike cells and also optimized for the obtainment of hepatic cells cultured on acellular matrix derived from liver decellularization process. It is well-known that TGF-beta exert an effect of extracellular matrix (ECM) remodelling and collagen deposition, nevertheless is not known the effect that ECM induces on cells during fibrotic process. In this context decellularized extracellular matrix was also derived from healthy donor or fibrotic patients in order to analyse the effect of ECM composition on secretion of endogenous factors mediated by hepatic cell, revealing an effect on modulating TGF-beta production.

Abstract (italian)

Il Trasforming Growth factor beta (TGF-beta) è una citochina multifunzionale che controlla differenti processi cellulari tra cui proliferazione, differenziamento, migrazione e apoptosi. Sebbene il TGF-beta controlli una varietà di processi biologici, la via di segnale è espressa in una maniera relativamente semplice. Il TGF-beta è secreto come complesso latente e richiede un'attivazione enzimatica per legare il suo recettore e mediare il segnale intracellulare. Il controllo temporale della via di segnale è essenziale per regolare il processo di embriogenesi e il mantenimento dell'omeostasi nel tessuto maturo. Disregolazioni della via di segnale sono associate a condizioni patologiche come fibrosi e carcinogenesi. La prima parte di questo lavoro è focalizzata sulla via di segnale di TGF-beta.
Esponendo cellule integrate in una piattaforma microfluidica ad amministrazioni multiple e periodiche di TGF-beta, è stato identificato come differenti stimolazioni con TGF -beta sono associate a differenti dinamiche della via di segnale ed espressione di geni target. In particolare è stata focalizzata l' attenzione sulla proteina SMAD7, responsabile dell' inattivazione della via di segnale ed è stato dimostrato che l' espressione di SMAD7 è indipendente dalla frequenza di stimolazione con TGF-beta. Differenti risultati clinicamente rilevanti possono essere ottenuti controllando l'attivazione, durata e intensità  della via di segnale a livello intracellulare ed extracellulare, quindi, utilizzare come bersaglio la via di segnale di TGF-beta nel trattamento farmacologico di patologie epatiche costituisce un approccio innovativo. Nella seconda parte di questo lavoro, grazie all'utilizzo della tecnologia microfluidica è stato prodotto un dispositivo per analizzare alcuni aspetti del processo di fibrosi epatica in vitro. In questa piattaforma microfluidica il microambiente promuove l'accumulo di fattori profibrotici o antifibrotici in risposta a stimoli profibrotici o antifibrotici, secreti da differenti tipi cellulari, coinvolti nel processo di fibrosi epatica. E' stata valutata la produzione ,
attivazione e funzione di TGF-beta endogeno da cellule del microambiente epatico, con particolare attenzione su cellule stellate e macrofagi, che sono rispettivamente responsabili del processo fibrotico e infiammatorio. E' stato inoltre analizzato lo specifico effetto della glicoproteina di matrice Trombospondina-1 nell'attivazione del TGF-beta in forma latente. La Trombospondina-1 è incrementata nel siero di pazienti fibrotici, per questa ragione è stato valutato l'effetto della trombospondina-1 nell' attivazione del TGF-beta autocrino secreto dalle cellule stellate epatiche. E' stata inoltre analizzata la produzione di collagene mediata da TGF-beta in cellule epatiche e cellule stellate, rilevando come queste ultime producano collagene in risposta a TGF-beta endogeno o esogeno. La terza parte del lavoro si è focalizzata sullo sviluppo di un modello di epatocita umano derivato da cellule staminali umane pluripotenti indotte utilizzando piattaforme microfluidiche. Il protocollo di differenziamento è stato utilizzato per ottenere cellule epatiche funzionali e ottimizzato per l'ottenimento di cellule epatiche in cultura su matrici acellulari derivate dal processo di decellularizzazione di fegato. E' risaputo che il TGF-beta induce un effetto di rimodellamento della matrice extracellulare e la deposizione di collagene, tuttavia non è noto l'effetto che la matrice extracellulare induce sulle cellule durante il processo fibrotico. In tale contesto matrici decellularizzate di fegato umano sono state ottenute da donatore sano o paziente fibrotico per analizzare l'effetto della differente composizione della matrice extracellulare sulla secrezione di fattori endogeni mediata da cellule epatiche, rilevando un effetto sulla modulazione della produzione di TGF-beta.

EPrint type:Ph.D. thesis
Tutor:Bolego, Chiara
Supervisor:Elvassore, Nicola
Ph.D. course:Ciclo 30 > Corsi 30 > SCIENZE FARMACOLOGICHE
Data di deposito della tesi:16 February 2018
Anno di Pubblicazione:15 January 2018
Key Words:microfluidics, TGF-beta, autocrine, paracrine, real-time, fibrosis.
Settori scientifico-disciplinari MIUR:Area 05 - Scienze biologiche > BIO/14 Farmacologia
Struttura di riferimento:Dipartimenti > Dipartimento di Scienze del Farmaco
Codice ID:11145
Depositato il:25 Oct 2018 16:00
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